Nuclear import of HIV-1 is a key step in the viral life cycle that to date has not been exploited as a target for anti-retroviral therapy. Accordingly, we have identified discontinuous charged-clusters of basic amino acids in the carboxyl-terminus of the HIV-1 integrase protein that act together to affect a powerful nuclear localization signal (HIV-1 IN NLS) in the context of both the native integrase protein and in infection of primary cell culture. Indeed, our analysis of HIV-1 molecular clones containing these mutations demonstrates that the HIV-1 IN NLS is an absolute requirement for viral replication in several cell types known to support HIV infection including human macrophages and activated peripheral blood mononuclear cells. The fact that our mutants are defective in dividing as well as non-diving cells is concordant with recent data indicating that nuclear transport of the HIV-1 genome is independent of mitotic nuclear disassembly in cycling cells (Katz et al., J. Virol. 77: 13412-13417). Although the requisite cellular factors that coordinate integrase-mediated viral nuclear transport remain largely unknown, several candidate factors have recently been put forward as likely contributors to this process. For example, EED (embryonic ectoderm development), a polycomb-group protein, co-localizes with integrase in the vicinity of the nuclear pore complex and binds integrase in a region that overlaps with the domain we have identified as having NLS activity (Violot et al., J. Virol. 77:12507-22). Intriguingly, specific binding between integrase and EED can be competed with phage that display peptide segments that map to the same set of charged-cluster amino acid residues that we have identified as critical to integrase NLS function in vivo. In this proposal we seek to clarify the contribution made by EED, and other cellular factors that have yet to be identified to HIV-1 integrase-mediated nuclear transport. The integrase NLS mutants we have described will be critical tools for these analyses. These studies, in turn will provide the rationale for development of assays in which the interaction between HIV-1 integrase and the requisite cellular co-factor(s) can be examined and potentially inhibited. We hypothesize that blockade of the interactions between integrase and its relevant cellular partners will result in the abrogation of the viral life cycle at a step preceding integration. Therefore, small molecule (or peptido-mimetic) inhibitors of HIV-1 integrase-mediated nuclear import would represent a new class of antiviral agents to curtail HIV infection.